Measurement of Brain CDP-Diacylglycerol Synthase 1 Enzyme and Uses Thereof

ABSTRACT

Provided herein are methods of diagnosing depressive disorders in a subject by comparing a CDP-diacylglycerol synthase 1 enzyme activity or expression level to enzyme activity or expression level of CDP-diacylglycerol synthase 1 in a control subject. Also herein are methods of predicting therapeutic efficacy of an antidepressant drug in a subject with depressive disorders by monitoring enzyme activity or expression level of CDP-diacylglycerol synthase 1 in the subject. Further provided herein are methods of identifying a compound effective to treat or alleviate the symptoms of depression by monitoring enzyme activity or expression level of CDP-diacylglycerol synthase 1 in a tissue. Further provided are kits for diagnosing depressive disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This continuation in part patent application claims benefit of priorityunder 35 U.S.C. §120 of pending divisional application U.S. Ser. No.12/804,534, filed Jul. 23, 2010 which claims benefit of priority under35 U.S.C. §120 of nonprovisional application U.S. Ser. No. 11/891,210,filed Aug. 9, 2007, now U.S. Pat. No. 7,763,603 which claims benefit ofpriority under 35 U.S.C. §119(e) of provisional application U.S. Ser.No. 60/836,904, filed Aug. 10, 2006, now abandoned, the entirety of bothof which are hereby incorporated by reference.

FEDERAL FUNDING LEGEND

This invention was produced in part using funds obtained through theNational Institutes of Health NIDA Grant No. DA017614. Consequently, thefederal government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of psychiatricmedicine and pharmacology. More specifically, the present inventionprovides methods to diagnosis and prognosis of depressive disorders bytargeting specific signaling molecules.

2. Description of the Related Art

Major depression is a serious mood disorder that annually afflictsmillions of people worldwide (1-3). Despite years of research, thebiological causes and pathological mechanisms of major depressivedisorder are unclear. Further, while treatments are available formanaging the disease symptoms, efforts to develop safer and moreeffective medications are hampered because the mechanism of action ofantidepressants is not well understood (4-5).

It is well known, however, that antidepressants with differing chemicaland clinical properties can increase the synaptic levels of theneurotransmitters, i.e., serotonin, norepinephrine, and/or dopamine, indiscrete brain regions (6-7). The monoamine transmitters may thenactivate their cognate postsynaptic receptors and modulate theactivities of downstream signaling cascades to possibly produce theantidepressive effect. It remains to be clarified, however, which amongthe numerous postsynaptic receptors and signaling components may beinvolved in the mode of action of antidepressants (5, 8-9).

Receptors for the monoamine neurotransmitters are coupled to diversesignaling pathways, including adenylyl cyclase, phospholipases, and MAPkinase pathways (10-13). Aspects of each of these signaling systems havebeen investigated as potential downstream targets of antidepressivemechanisms (8, 14-15). As examples, acute or chronic treatment withvarious antidepressant compounds can lead to changes in basal ordrug-induced activities of brain adenylyl cyclase (16-19), phospholipaseA2, CREB, inositol phosphates (IPs), phosphoinositide-specificphospholipase C (PLC), phosphatidylinositides, protein kinase C (PKC),extracellular signal regulated kinase, ion channels, neurotrophins, andneuropeptides. Antidepressants can also enhance neurogenesis, modulateneuronal excitability, and alter the gene expression of varioussignaling components including neurotransmitter transporters, receptors,transducers, and effectors (39-46). While these observations indicatethat changes in postsynaptic signaling cascades may constitute anintegral component in the mechanisms that underlie depression or itstreatment with antidepressant medications, no signaling cascade has beenidentified that explains the functional and clinical data.

The depression or antidepressant-related phosphoinositide observationshave been corroborated by clinical studies showing that depressedpersons have reduced cortical levels of the phosphoinositide precursormyo-inositol (47-48). Moreover, oral ingestion of pharmacological dosesof myo-inositol may elicit antidepressive responses in rodents andenhance the recovery of clinically depressed patients (49-51).Consistent with these findings, chronic administration of antidepressantagents has been associated with increased levels of phosphatidylinositol(PI), phosphatidylinositol phosphate (PIP), and phosphatidylinositolbisphosphate (PIP2) in human platelets (24,27). These observationssupport the notion that alterations in the phosphoinositide signalingpathway may be implicated in the pathophysiology of depression and/orthe mode of action of antidepressant agents (5,26,52).

Several studies have hinted at links between the phosphoinositidesystem, PKC activity, and depression (28-30). However, these studieshave not assessed the status of diacylglycerol production or metabolismas a potential target of disease pathology or pharmacological treatment.Diacylglycerol signaling is important as it is the endogenous regulatorof PKC activity (Nishizuka, 1992). Among PLC-coupled receptors, however,significant differences exist in the ability of receptor activation togenerate diacylglycerol, relative to IP, from receptor-mediatedphospholipid hydrolysis (53-54).

It is recognized, therefore, to the extent that PI signaling or PKCactivity may be involved in antidepressant drug action, that there is asignificant need in the art for improvements in the area of treatingdepression with antidepressant agents that target cellularCDP-diacylglycerol. Specifically, the present invention is deficient inmethods of screening for novel compounds that increase cellularCDP-diacylglycerol and methods of diagnosing and treating depressionusing the same. The present invention fulfills this long-standing needand desire in the art.

SUMMARY OF THE INVENTION

It is an object of the present invention to demonstrate that the proteinand/or mRNA levels of two CDP-diacylglycerol synthase (CDS)isoforms—CDS1 and CDS2—are altered in postmortem forebrain tissues ofdepression subjects.

It is a further object of the present invention to demonstrate thatthere are measurable levels of CDS isozymes in circulating peripheralblood.

The present invention is directed, in part, to a method of diagnosing adepressive disorder in a test subject. The method comprises determiningCDP-diacylglycerol synthase 1 enzyme activity or expression level in thetest subject and comparing the enzyme activity or CDP-diacylglycerolsynthase 1 expression level to enzyme activity or expression level ofCDP-diacylglycerol synthase 1 in a control subject. A statisticallylower enzyme activity or expression level in the test subject indicatesthe test subject has a depressive disorder.

The present invention is directed to a related method of predictingtherapeutic efficacy of an antidepressant drug in a subject with adepressive disorder. The method comprises determining CDP-diacylglycerolsynthase 1 enzyme activity or expression level of CDP-diacylglycerolsynthase 1 in the subject, administering an antidepressant drug to thesubject and monitoring CDP-diacylglycerol synthase 1 enzyme activity orexpression level of CDP-diacylglycerol synthase 1 in the subject afteradministration of the antidepressant drug to the subject. Astatistically higher CDP-diacylglycerol synthase 1 enzyme activity orCDP-diacylglycerol synthase 1 expression level after administration ofthe antidepressant drug to the subject indicates that the antidepressantdrug has therapeutic efficacy for the subject.

The present invention is directed further yet to a method of identifyinga compound effective to treat or alleviate the symptoms of depression.The method comprises determining CDP-diacylglycerol synthase 1 enzymeactivity or expression level of CDP-diacylglycerol synthase 1 in atissue, contacting the tissue with a potential antidepressant compoundand determining the level of CDP-diacylglycerol synthase 1 enzymeactivity or expression level of CDP-diacylglycerol synthase 1 in thetissue after the contact. A statistically higher CDP-diacylglycerolsynthase 1 enzyme activity or CDP-diacylglycerol synthase 1 expressionlevel in the tissue after treatment with the compound indicates that thecompound has an antidepressant effect.

The present invention is directed further still to a kit for diagnosinga depressive disorder. The kit comprises an antibody toCDP-diacylglycerol synthase 1 protein, an antibody to CDP-diacylglycerolsynthase 2 protein and instructions for measurement of the proteins forthe diagnosis of the depressive disorder.

The present invention is directed further still to a kit for diagnosinga depressive disorder. The kit comprises forward and reverse primers forCDP-diacylglycerol synthase 1 gene, a probe that hybridizes withCDP-diacylglycerol synthase 1 gene, forward and reverse primers forCDP-diacylglycerol synthase 2 gene, a probe that hybridizes withCDP-diacylglycerol synthase 2 gene and instructions for measurement ofsaid genes for the diagnosis of said depressive disorder.

Other and further aspects, features and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention are briefly summarized. The above may be betterunderstood by reference to certain embodiments thereof which areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted; however, that the appended drawingsillustrate preferred embodiments of the invention and therefore are notto be considered limiting in their scope.

FIGS. 1A-1C shows High and specific immunoreactivity of rabbitantibodies raised against the human CDS1 and CDS2 proteins usingepitopes designed to cross-react with the rat CDS1 and CDS2,respectively. Rabbit anti-human/rat preimmune sera (Pre), first-bleed(B1) second-bleed (B2) and protein A-purified second-bleed (B2p)antisera were tested against protein extracts from rat cerebellum (Cb)or testes (T) or against extracts of HEK293 cells transfected with empty(HO), CDS1 (H1) or CDS2 (H2) plasmids. Antisera (B1 or B2) fromcds2-immunized rabbits showed strong reactions with cerebellar ortesticular protein extracts (FIG. 1A), while antisera (B2) fromcds1-immunized rabbits showed high reactivity with testicular extractsand moderate reactivity with cerebellar extracts (FIG. 1B). Wild typeand cds2-transfected HEK293 cells showed substantial reaction to proteinA-purified cds1 antibodies, indicating the presence of wild type cds1 inthese cells (FIG. 1C). Cells transfected with cds1, however, gaveoverwhelming reactivity to the cds1 antibody. Importantly, the cds1 andcds2 antibody reactivities were located at approximately the expected Mrof 65 kD and 51 kD, respectively.

FIGS. 2A-2B show CDS1 mRNA (FIG. 2A) and protein levels (FIG. 2B) inpostmortem frontal cortex Area BA9 samples from normal control (nodepression or antidepressant (AD) drug exposure) and depression (Dep)subjects without (No-AD) or with AD (+AD) medication. Depressionsubjects showed significantly lower levels of CDS1 message and protein,while those depression subjects that had taken (any) antidepressantmedication showed intermediate levels of CDS1 mRNA and protein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “a” or “an”, when used in conjunction with theterm “comprising” in the claims and/or the specification may mean “one”,but it is also consistent with the meaning of “one or more”, “at leastone”, and “one or more than one”. Some embodiments of the invention mayconsist of or consist essentially of one or more elements, method steps,and/or methods of the invention. It is contemplated that any method,compound, drug, or composition described herein can be implemented withrespect to any other method, compound, drug, or composition describedherein.

As used herein, the term “or” in the claims is used to mean “and/or”unless explicitly indicated to refer to alternatives only or thealternatives are mutually exclusive, although the disclosure supports adefinition that refers to only alternatives and “and/or”.

As used herein, the term “antidepressant drug or agent” refers to knowncompounds exhibiting an antidepressive effect or antidepressant effecton a subject having depression or a depressive disorder or mooddisorder. Antidepressants may be, but not limited to, the tricyclicantidepressants, e.g., desipramine and imipramine, the selectiveserotonin reuptake inhibitors, e.g., fluoxetine and paroxetine, theatypical antidepressants, e.g., maprotiline and nomifensine, or de novocompounds SKF83959 or6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine(55) and SKF38393 or(+/−)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (56).

As used herein, the term “potential antidepressant compound” or“screened antidepressant compound” refers to a compound having, althoughnot limited to, a similarity in structure, such as a derivative oranalog, to a known antidepressant drug or agent” and/or a therapeuticability to at least increase accumulation of cellular CDP-diacyglyceroland inositol phosphate and enhance synthesis of inositol phospholipidsin brain tissue and/or blood cells.

As used herein, the term “structural derivative” refers to a change tothe structure of an original compound that conserves the functionalaspects, i.e., biological activity, efficacy, and the like, of theoriginal compound. For example, an original compound may be SKF83959 anda structural derivative may include an addition and/or a modification ofthe original benzazepine structure, such as changing halogensubstituents, oxidation state, hydration, salt counterions, and thelike.

As used herein, the term “CDP-DG/IP ratio” refers to the relative valueof an index which is the quotient of a level of cellularCDP-diacylglycerol divided by a level of inositol phosphate in a tissueor cell or tissue cultures thereof, e.g., a mammalian brain tissue orblood platelets, a human brain tissue or blood platelets, or other cellsor tissues having the ability to metabolize CDP-diacylglycerol andphosphoinositides. Levels may be determined, including but are notlimited to, using molar concentration or a radiolabel.

As used herein, the term “contacting” refers to any suitable method ofbringing an antidepressant drug or agent or potential antidepressantcompound into contact with a tissue or cell, e.g., a mammalian braintissue or blood platelets or other blood cells, a human brain tissue,blood platelets or other blood cells, or other cells or tissues havingthe ability to metabolize CDP-diacylglycerol and phosphoinositides. Invitro or ex vivo this is achieved by exposing the tissue in a cell ortissue culture to the anti-depressive agent or potential antidepressivecompound in a suitable medium. For in vivo applications, anyappropriately known method of administration is suitable.

As used herein, the terms “effective amount” or “pharmacologicallyeffective amount” or “therapeutically effective amount” areinterchangeable and refer to an amount that results in an improvement orremediation of the symptoms of the depressive disorder or condition. Theamount is sufficient to detectably and repeatedly to ameliorate, reduce,minimize, or limit the extent of the depressive disorder or condition orthe symptoms thereof. Those of skill in the art understand that theeffective amount may improve the patient's or subject's condition, butmay not be a complete cure of the depressive disorder and/or condition.

As used herein, the term “depressive disorder” or “mood disorder” refersto clinical depression, major depression, unipolar depression, reactivedepression, endogenous depression, dysthymia, or bipolar disorder. Majordepression, which is also known as clinical depression or unipolardepression, is a mood disorder in which feelings of sadness, loss,anger, or frustration interfere with everyday life for weeks or longer.Bipolar disorder, also known as manic-depressive disorder, is a mooddisorder that causes radical emotional changes and mood swings, frommanic, restless highs to depressive, listless lows. Reactive depressionis a usually transient depression that is precipitated by a stressfullife event or other environmental factor. Endogenous depression iscaused by an intrinsic biological or somatic process rather than anenvironmental influence, in contrast to a reactive depression. Dysthymicdepression is a mood disorder with mild and chronic depressive symptomsthat are present most of the day, more days than not, for a period of atleast two years.

As used herein, the term “subject” refers to any target of thetreatment, preferably a mammal, more preferably a human.

In one embodiment of the present invention, there is provided a methodof identifying a compound effective to treat or alleviate the symptomsof depression, comprising contacting a tissue having aCDP-diacylglycerol and phosphoinositides metabolic activity with apotential antidepressant compound; determining a level ofCDP-diacylglycerol (CDP-DG) and a level of inositide (inositol phosphateand/or phosphoinositide) in the tissue after contact therewith; andcomparing an index value that is a ratio of CDP-diacylglycerol toinositide in test tissue to a control index value, wherein a higherCDP-DG/Inositide index value in tissue treated with the compoundindicates the potential compound has an antidepressant effect.

Further to this embodiment, the method comprises designing the potentialantidepressive compound prior to screening, where the design is based onthe structure of a tricyclic antidepressant, a selective serotoninreuptake inhibitor or an atypical antidepressant or on a structure of acompound structurally dissimilar thereto exhibiting an antidepressanteffect or synthesizing a potential compound de novo. Examples of atricyclic antidepressant is desipramine or imipramine. Examples of aselective serotonin reuptake inhibitor are fluoxetine or paroxetine.Examples of an atypical antidepressant are maprotiline or nomifensine.Other atypical antidepressants may be SKF83959, or SKF38393 or astructural derivative thereof. In another further embodiment the methodcomprises treating a subject having a depressive disorder with thecompound screened by the method.

In another embodiment of the present invention, there is provided acompound screened by the method described supra. In a related embodimentthere is provided a synthetic antidepressant compound effective toincrease production of CDP-diacylglycerol and synthesis of inositide ina depression-relevant brain tissue or blood tissue upon contacttherewith. In both embodiments, the screened compound and the syntheticantidepressant compound may be an analog or derivative of a tricyclicantidepressant, a selective serotonin reuptake inhibitor or an atypicalantidepressant. Examples of these antidepressants are described supra.In another related embodiment there is provided a synthetic compoundeffective to increase CDP-diacylglycerol synthase activity in adepression-relevant brain tissue, blood cells or other body tissue uponcontact therewith.

In yet another embodiment of the present invention, there is provided amethod of treating a depressive disorder in a subject, comprisingadministering one or more of the screened compounds described supra tothe subject, thereby treating the antidepressive disorder. Further tothis embodiment the method comprises administering one or more otherknown antidepressant drugs or other known compounds effective toincrease an CDP-DG/Inositide index. The antidepressant drugs may be atricyclic antidepressant, a selective serotonin reuptake inhibitor or anatypical antidepressant with specific examples thereof as describedsupra. The other known compound is SKF83959 or SKF38393 or a structuralderivative thereof. Examples of the depressive disorder include but arenot limited major depression, unipolar depression, bipolar depression,reactive depression, endogenous depression or dysthymic disorder.

In another further embodiment the method comprises diagnosing thedepressive disorder in a subject prior to treatment thereof. In thisfurther embodiment diagnosing the depressive disorder comprisesdetermining a level of CDP-diacylglycerol and a level of inositide inthe subject; and comparing an index value that is a ratio ofCDP-diacylglycerol to inositide in the subject to a control index value,wherein a lower CDP-DG/Inositide index value indicates the subject has adepressive disorder.

In yet another further embodiment, the method comprises determining acombination of the screened antidepressive compounds, the otherantidepressant drugs or a combination thereof having maximum therapeuticefficacy against the depressive disorder. Determining the combination ofantidepressant drugs comprises administering a first selectedcombination of antidepressants to the subject; determining a first indexvalue that is a level of CDP-diacylglycerol (CDP-DG) and of inositide inthe subject after administration of the antidepressant combination; andcomparing the first index value to the CDP-DG/Inositide index values ofsubsequently and individually administered combinations of otherantidepressants; wherein the combination having the highest CDP-DG indexcorrelates to a maximum therapeutic efficacy.

In a related embodiment, there is provided a method of treatingdepression in a subject, comprising administering aCDP-diacylglycerol-increasing amount of a compound to the subject, wherethe compound increases CDP-diacylglycerol metabolism in the subject,thereby treating the depression. Representative examples of the compoundare maprotiline, nomifensine, SKF83959, SKF38393, or a structuralderivatives thereof.

In yet another embodiment, there is provided a method of diagnosing adepressive disorder in a subject, comprising determining a basalCDP-DG/Inositide index that is the ratio of a level ofCDP-diacylglycerol to a level of inositide in the subject; and comparingthe basal CDP-DG/Inositide index to a control CDP-Inositide index, wherea lower basal CDP-DG/Inositide index indicates the subject has adepressive disorder. In this related embodiment the diagnosis may bepredictive of the onset of a depressive disorder. The depressivedisorder may be as described infra.

In still another embodiment of the present invention there is providedmethod of predicting therapeutic efficacy of an antidepressant drugregimen in a subject having a depressive disorder, comprisingadministering a first selected drug regimen that is a combination ofantidepressant drugs to the subject; determining a firstCDP-DG/Inositide index value that is the ratio of a level ofCDP-diacylglycerol (CDP-DG) to a level of inositide in the subject afteradministration; comparing the first index value to the CDP-DG/Inositideindex values of subsequently and individually administered combinationsof other selected antidepressants; wherein an ordering of the relativeindex values correlates to therapeutic efficacy of the drug regimen. Inthis embodiment, the combination of antidepressant drugs may comprise atricyclic antidepressant(s), a selective serotonin reuptake inhibitor(s)or an atypical antidepressant(s) or derivatives or analogs thereof or anovel synthetic antidepressant compound designed de novo that increasesproduction of CDP-diacylglycerol and synthesis of inositide in adepression-relevant brain tissue or blood cells or other body tissue.The examples of a tricyclic antidepressant, a selective serotoninreuptake inhibitor and an atypical antidepressant are as describedsupra. Also, the depressive disorder may be as described supra.

The present invention demonstrates that antidepressants belonging todiverse chemical and pharmacological classes acutely increase theformation of CDP-diacylglycerol (CDP-DG), a metabolic derivative ofdiacylglycerol, which effect may translate to enhanced resynthesis ofthe phosphatidylinositides. Phosphatidylinositides are metabolicallyused either as substrates for PLC or as precursors to thephosphatidylinositol-3-kinase (PI-3-K)/Akt signaling cascade. It iscontemplated, therefore, that an acute molecular action ofantidepressant agents that facilitates the conservation orsupplementation of cellular phosphatidylinositides may contribute to thetherapeutic mechanism of these medications in depression-relevant brainregions.

It is also contemplated that known antidepressants may exert tandemneurochemical effects by increasing synaptic monoamine concentrationsand by producing phosphoinositide substrates used in 5HT2 receptorsignaling. This combination of actions may constitute the mechanism ofat least the acute behavioral effects of antidepressant medications and,thereby, may implicate aberrant phospholipid signaling in theneuropathology of depressive disorder.

Thus, provided herein is a method of screening for potentialantidepressant compounds. Potential antidepressant agents aredistinguished from other compounds that enhance phosphoinositidesignaling by the relative value of the CDP-DG/Inositide index. Anincrease in the CDP-DG/Inositide index compared to a control index isindicative that the potential antidepressant compound may exhibitantidepressant effects by at least increasing cellularCDP-diacylglycerol in a subject with a depressive or mood disorder.Cellular levels of CDP-diacylglycerol and inositide may be determined inbrain tissue, blood platelets or other blood cells, cultured cells orcombination of tissues in the presence and absence of potentialantidepressant compounds using assay methods known and standard in theart.

As such, the antidepressant compounds identified by the screening methodalso are provided. It is contemplated that these screened antidepressantcompounds may be derivatives or analogs of known antidepressant drugs oragents as described herein. Alternatively, the antidepressant compoundsmay have a novel synthetic structure designed de novo using standardmethods of chemical design known in the art, for example, but notlimited to, computer aided drug design.

Compositions, Pharmaceutical Formulations and Methods of Treating

The present invention also contemplates therapeutic or treatment methodsemploying compositions comprising the screened antidepressant compoundsdisclosed herein, that is, compositions comprising the knownantidepressant drugs and/or the screened antidepressant compoundsprovided herein. Preferably, these compositions include pharmaceuticalcompositions comprising a therapeutically effective amount of one ormore of the active compounds or substances along with conventionalnon-toxic, physiologically or pharmaceutically acceptable carriers orvehicles suitable for the method of administration.

Treatment methods will involve treating an individual with an effectiveamount of a composition containing the screened antidepressant compoundand/or known antidepressant drug or related compounds thereof. Morespecifically, it is envisioned that the treatment with theantidepressant compounds and/or antidepressant drugs orrelated-compounds thereof will increase production of CDP-diacylglyceroland synthesis of inositol phospholipids in depression-relevant braintissues or blood platelets or other blood cells to produce a beneficialresult in a depressive or mood disorder.

Thus, methods of diagnosing a depressive disorder in a subject,preferably a human, are provided. Brain, peripheral tissue, platelet orother blood cell levels of CDP-diacylglycerol are measured to aiddiagnosis of an active or impending depressive episode. A determinationof an abnormal CDP-diacylglycerol signaling in the subject may beindicative of a depressive or mood disorder. Therefore, theCDP-DG/Inositide index may be a useful diagnostic tool in the diagnosisof a depressive or mood disorder.

In addition, methods of treating a depressive or mood disorder in asubject are provided. A pharmacologically effective or therapeuticallyeffective amount of one or more of the screened antidepressive compoundsdescribed herein or a pharmaceutical composition comprising the same isadministered to the subject. Alternatively, treatment may comprise acombination of the screened antidepressive compound(s) and one or moreknown antidepressants or pharmaceutical compositions thereof. As such,treatment by, for example, but not limited to, atypical antidepressantsor structural derivatives thereof provide a therapeutic effect of atleast increasing CDP-diacylglycerol metabolism.

Further provided is a method of predicting the potential effectivenessof antidepressant combinations for various patients based on the basalCDP-DG/Inositide ratio of each patient. An increase in theCDP-DG/Inositide index compared to the basel index of current or novelantidepressant compounds may be predictive of antidepressant effects ina subject. A successful regimen may comprise a combination of the knowntricyclic antidepressants, the known selective serotonin reuptakeinhibitors and the known atypical antidepressants and/or the novelantidepressant compounds screened as described herein.

In still another embodiment of the present invention, there is provideda method of diagnosing a depressive disorder in a test subject. Themethod comprises determining CDP-diacylglycerol synthase 1 enzymeactivity or CDP-diacylglycerol synthase 1 expression level in the testsubject and comparing the enzyme activity or expression level toCDP-diacylglycerol synthase 1 enzyme activity or expression level ofCDP-diacylglycerol synthase 1 in a control subject. A statisticallylower enzyme activity or expression level in the test subject indicatesthe test subject has a depressive disorder. In this embodiment, theexpression level is typically determined by measuring aCDP-diacylglycerol synthase 1 protein, a ratio of a CDP-diacylglycerolsynthase 1 protein to a CDP-diacylglycerol synthase 2 protein, aCDP-diacylglycerol synthase 1 mRNA or a ratio of a CDP-diacylglycerolsynthase 1 mRNA to a CDP-diacylglycerol synthase 2 mRNA. The enzymeactivity or expression level of CDP-diacylglycerol synthase is typicallydetermined in blood, brain tissue, testis tissue, eye tissue or smoothmuscle tissue. M Examples of the depressive disorder include but are notlimited to major depression, unipolar depression, bipolar depression,reactive depression, endogenous depression, or dysthymic disorder.

Further to this embodiment, a CDP-diacylglycerol synthase 1 proteinlevel in the test subject that is about 75% or lower than aCDP-diacylglycerol synthase 1 protein level in the control subjecttypically indicates that the test subject has a depressive disorder. ACDP-diacylglycerol synthase 1 mRNA level in the test subject that isabout 68% or lower than a CDP-diacylglycerol synthase 1 mRNA level inthe control subject typically indicates that the test subject has adepressive disorder. A ratio of a CDP-diacylglycerol synthase 1 mRNA toa CDP-diacylglycerol synthase 2 mRNA in the test subject that is about73% or lower than a ratio of a CDP-diacylglycerol synthase 1 mRNA to aCDP-diacylglycerol synthase 2 mRNA in the control subject typicallyindicates that the test subject has a depressive disorder.

In still another embodiment of the present invention, there is provideda method of predicting therapeutic efficacy of an antidepressant drug ina subject with a depressive disorder. The method comprises determiningCDP-diacylglycerol synthase 1 enzyme activity or expression level ofCDP-diacylglycerol synthase 1 in the subject, administering anantidepressant drug to the subject and monitoring enzyme activity orexpression level of CDP-diacylglycerol synthase 1 in the subject afteradministration of the antidepressant drug to the subject. Astatistically higher enzyme activity or expression level afteradministration of the antidepressant drug to the subject indicates thatthe antidepressant drug has therapeutic efficacy for the subject.

In this embodiment, examples of the antidepressant drug include but arenot limited to a tricyclic antidepressant, a selective serotoninreuptake inhibitor, an atypical antidepressant or a syntheticantidepressant compound that increases an enzyme activity or expressionlevel of a CDP-diacylglycerol synthase 1 in a depression-relevant braintissue or blood platelets. A representative tricyclic antidepressant isdesipramine or imipramine and a representative selective serotoninreuptake inhibitor is fluoxetine or paroxetine. Examples of thedepressive disorder include but are not limited to major depression,unipolar depression, bipolar depression, reactive depression, endogenousdepression, or dysthymic disorder. Further to this embodiment, theexpression level is typically determined by measuring aCDP-diacylglycerol synthase 1 protein, a ratio of a CDP-diacylglycerolsynthase 1 protein to a CDP-diacylglycerol synthase 2 protein, aCDP-diacylglycerol synthase 1 mRNA or a ratio of a CDP-diacylglycerolsynthase 1 mRNA to a CDP-diacylglycerol synthase 2 mRNA. TheCDP-diacylglycerol synthase 1 enzyme activity or expression level ofCDPdiacylglycerol synthase is typically determined in blood, braintissue, testis tissue, eye tissue or smooth muscle tissue.

In still another embodiment of the present invention, there is provideda method of identifying a compound effective to treat or alleviate thesymptoms of depression. The method comprises determining level of enzymeactivity or expression level of CDP-diacylglycerol synthase 1 in atissue, contacting the tissue with a potential antidepressant compoundand determining level of CDP-diacylglycerol synthase 1 enzyme activityor expression level of CDP-diacylglycerol synthase 1 in the tissue afterthe contact. A statistically higher enzyme activity or expression levelin the tissue after treatment with the compound indicates that thecompound has an antidepressant effect. The present invention is directedfurther still to a kit for diagnosing a depressive disorder. The kitcomprises an antibody to CDP-diacylglycerol synthase 1 protein, anantibody to CDP-diacylglycerol synthase 2 protein and instructions formeasurement of the proteins for the diagnosis of the depressivedisorder.

The present invention is directed further still to a kit for diagnosinga depressive disorder. The kit comprises forward and reverse primers forCDP-diacylglycerol synthase 1 gene, a probe that hybridizes withCDP-diacylglycerol synthase 1 gene, forward and reverse primers forCDP-diacylglycerol synthase 2 gene, a probe that hybridizes withCDP-diacylglycerol synthase 2 gene and instructions for measurement ofsaid genes for the diagnosis of said depressive disorder.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1 Animals

Male Sprague-Dawley rats, weighing between 225 g-300 g, were obtainedfrom Zivic Laboratories (Zelienople, Pa.) and housed inclimate-controlled facilities with a 12-h light/dark cycle for at least3 days before use. The animals were caged in groups of three and allowedfree access to food and water.

Drugs and Chemicals

Antidepressant compounds and buffer reagents were purchased fromSigma-Aldrich (St. Louis, Mo.). SKF38393 was from the NIMH ChemicalSynthesis Program (NIMH, Bethesda, US). Nomifensine was first dissolvedin 0.2% tartaric acid and SKF38393 in distilled water before either drugwas diluted to use concentrations in assay buffer. Other drugs wereprepared fresh in HEPES bicarbonate assay buffer (HBB) (57). Eachexperiment was performed on multiple occasions using fresh preparationsof drugs. Protein was assayed by the Bradford method using BioRadprotein assay reagents (BioRad, Hercules, Calif.).

Measurement of CDP-diacylglycerol Accumulation

Accumulation of CDP-diacylglycerol was measured in brain slicepreparations by taking advantage of the CTP-phosphatidate transferreaction (58-60). Briefly, male Sprague-Dawley rats weighing between 225and 300 g were rapidly decapitated and the brains removed and rinsed incalcium-free HBB (58,61). Brain regions of interest, including thehippocampus, prefrontal cortex and striatum, were quickly dissected outand 350 μm prisms prepared using a Mcllwain tissue chopper (61). Theslices were washed with calcium-free HBB and pre-incubated for 45minutes at 37° C. Slice aliquots of approximately 300 μg protein thenwere incubated with 1.5 μCi of 5-[³H]cytidine (20 Ci/mmol; AmericanRadiolabeled Chemicals, St. Louis, Mo.) in order to generate anendogenous pool of radiolabeled cytidine triphosphate (CTP) for feedinginto the CTP:phosphatidate transfer reaction (58).

Following addition of 5 mM LiCl, test drugs or buffer were added for atotal volume of 250 μl and incubation continued for 60 or 90 min asindicated. Reactions were terminated by addition of 1.5 mlchloroform-methanol-1M HCl (100:200:1). Formed lipids were extracted byliquid partitioning in chloroform followed by centrifugation at 1000×gfor 5 min to separate the liquid phases. Aliquots of the organic phasewere transferred quantitatively into scintillation vials, dried at roomtemperature and redissolved in Biosafe scintillation cocktail.Radioactivity in this lipid fraction was determined by liquidscintillation spectrometry, and corresponds to [³H]CDP-DG (54,58,62).

Measurement of Inositol Phospholipid Resynthesis

Brain tissues were prepared and were incubated as described above forassaying CDP-DG, except that 1.5 μCi of [³H]inositol (20 Ci/mmol;American Radiolabeled Chemicals, St. Louis, Mo.) was used instead of[³H]cytidine to label the slices. Following the labeling incubation,drugs were added and allowed to act for 60 or 90 min as indicated.Samples were extracted with chloroform-methanol-1M HCl (100:200:1),partitioned with chloroform into aqueous and organic phases, andaliquots of the organic phase dried and assessed for radioactivity thatcorresponded to the inositol phospholipids. It was not necessary toattempt to separate the multiple phosphorylated or isomeric forms ofthese phospholipids. Hence, the data potentially represent the mix ofphosphatidylinositol, phosphatidylinositol-4-phosphate, andphosphatidylinositol 4,5-bisphosphate in any of their positionalisomeric forms. Based on the levels of the phospholipids present at thestart of drug treatment, a subsequent decrease is seen as depletion,whereas an increase in the [³H]inositol-labeled pool of thephospholipids is considered to represent further phospholipid synthesisor resynthesis (59,63).

Measurement of Inositol Phosphate Accumulation

To measure the levels of IPs formed, tissues were treated exactly as inthe PI synthesis assays described herein, including the use of[³H]inositol for prelabeling of the PI pool. The 250 μl reactions wereterminated by mixing the samples with 1.5 ml of chloroform-methanol-1 MHCl (100:200:1). Following chloroform-mediated partitioning of theextracts as described (57), aliquots of the aqueous phase were analyzedfor the content of [³H]IPs by Dowex anion exchange chromatography(57,61). An IP fraction was collected from the eluate and the solutionconverted into a gel by use of Scintisafe Gel (Fisher Scientific,Pittsburgh, Pa.). The amounts of IP-associated radioactivity in thesamples were then measured by liquid scintillation spectrometry.

Forced Swim Test

The forced swim test (FST) was applied with some modifications. Ratswere transferred to the testing room between 9:00-10:00 AM and allowedat least an hour before being placed individually into translucentpolypropylene cylinders (46 cm tall×25 cm in diameter) containing 27 cmdepth of water maintained at 25° C. (64-65). After 15 min in the water,the rats were removed, toweled, and allowed to dry in a warm environmentbefore being returned to their home cage. This acclimation step wasrepeated after 24 h, with the exposure time reduced from 15 to 5 min.Preliminary testing showed that the double acclimation exposure producedmore consistent results among animals in each test group (lowervariability) than the conventional single acclimation.

During the second acclimation exposure, the duration of immobility wasrecorded for each animal. While the initial intent was to eliminateanimals that did not attain immobility within 5 min, in practice allanimals used in the present experiments passed this test at the secondacclimation session. To adjudge that a rat had become immobile, theanimal had to float passively in the water in a slightly hunched, butupright position, making only minimal movements necessary to keep itshead above the water (65-66).

On the third day when animals were to undergo experimental assessment,saline or the indicated antidepressant agents were administered i.p. at3 h and at 1 h before behavioral testing. The drugs were dissolved indistilled-deionized water and diluted in saline. Control subjectsreceived 0.9% saline. Drugs were freshly made before use and injected ina constant volume of 1 ml/kg except for fluoxetine which was given at avolume of 5 ml/kg. Neomycin was prepared as a solution in normal salineand injected into the tail vein 2 h prior to testing, that is, 1 hbetween the first and second administrations of the test drug. Thisapproach has been reported to be effective in inhibiting endogenousbrain PI metabolism for behavioral studies (67), although otherapproaches such as intracerebroventricular injection have also been used(68-69). Data were analyzed by one-way analysis of variance (ANOVA).Conclusions of mean differences were drawn when the calculated p-valueswere less than 0.05.

Brain Tissue Samples and Donor Demographics

The prefrontal cortex brain region was used, specifically Area BA 9 thatis implicated in major depression. Fresh frozen human frontal cortexsamples were obtained from the NICHD Brain and Tissue Bank at theUniversity of Maryland Baltimore (UMB Bank). The UMB Bank was selectedfor tissue procurement because of their growing catalog of frozen brainsamples from control and depression subjects, and the tissues areobtained through standardized dissection, handling, and preservationprotocols. To enhance homogeneity of samples, each tissue sample wasminced and the pieces mixed and then aliquots taken for downstreamanalysis. A set of 28 diagnosed depression cases having acceptablemedical history documentation was matched to a set of unaffectedcontrols based on sex, age (+/−3 yr), ethnicity, postmortem interval(PMI, +/−6 hr), and storage duration (+/−6 mo). Among the 28 pairs ofcontrol (Con) and depression (Dep) subjects that were matched, eachdiagnostic group consisted of 13 males and 15 females, 5 African and 23Caucasian. The mean age(±SD) of the groups was Con 34.3±17.6 and Dep34.4±17.3 yr, and the average PMI(±SD) is Con 17.8±7.2 and Dep 17.0±6.4hr. Among the Dep subjects, 18 had a medical history or autopsy recordof antidepressant medication use, and 6 had a record of othermedications which belong to pharmacologic classes not known tosignificantly affect CDP-diacylglycerol levels. Samples were coded toremain blind to the experimenter until data collection had completed.

Collection of Blood and RNA Extraction

Blood was collected from three healthy volunteers by finger prick usinga Actilance Universal contact-activated lancet (HTL-STREFA Inc.,Marietta, Ga.). Three drops of blood were transferred by capillary tubeinto EDTA-treated blood collection microtubes at room temperature andthe tube shaken to mix the blood with anticoagulant and preventclotting.

RNA and Protein Extraction

Approximately 100 mg of each brain tissue sample was homogenized andaliquots taken for RNA and protein extraction. In the case of the bloodsample, a 100-μl aliquot of whole blood was used. Total RNA wasextracted using SurePrep RNA purification kit (Fisher Scientific) usingthe manufacturer's protocol that is based on elution from RNA-bindingcolumns.

CDS protein was extracted from the same sample used for extraction ofRNA. The flowthrough from the RNA binding column together with the firstrinse of the column were collected into centrifuge tubes and spun at3000×g for 20 min to remove the red blood cells. The supernatantcontaining the blood plasma was collected as the crude protein extract.

Quantitative Realtime PCR for CDS1 and CDS2 mRNA Levels

The purified RNA sample extracted from brain or blood was quantifiedusing Nanodrop 2000c spectrophotometer (Thermo Scientific) and thepurity was checked by the 260/280 absorbance ratio. Quantitative RT-PCRanalysis was carried out using 100 ng of purified RNA. Verso One-stepqRT-PCR master mix (Thermo Fischer Scientific) and Solaris geneexpression assay system (ThermoScientific Dharmacon) were used. The mRNAexpression levels of CDS1, CDS2 and GAPDH were measured by qRT-PCR. Theprimers specific for these transcripts were, for CDS1-Forward primer:5′-CTGGTTTATCTTCAAGGTGGAAA-3′ (SEQ ID NO: 1), Reverse primer:5′-TTCACTTGGATGCCCAGAAC-3′ (SEQ ID NO: 2), Probe:5′-CTTCATGCTGATGCTTCTTG-3′ (SEQ ID NO: 3); for CDS2-Forward primer:5′-CCTATTTGAAGGAATGATCTGGTTC-3′ (SEQ ID NO: 4), Reverse primer:5′-GGCCATGATGTCATTACAG-3′ (SEQ ID NO: 5), Probe:5′-CCCATATCTTGTGTGATCT-3′ (SEQ ID NO: 6) and for GAPDH-Forward primer:5′-GCCTCAAGATCATCAGCAATG-3′ (SEQ ID NO: 7), Reverse primer:5′-CTTCCACGATACCAAAGTTGTC-3′ (SEQ ID NO: 8) and probe:5′-GCCAAGGTCATCCATGA-3′ (SEQ ID NO: 9). The cycling conditions in theEppendorff Mastercycler were 50° C. for 15 min, 95° C. for 15 min, and40 cycles of 95° C. for 15 sec and 60° C. for 60 sec. Cycle threshold(Ct) values were obtained from the Mastercycler software and therelative expression values of CDS1 and CDS2 were calculated by delta-Ctusing GAPDH as the reference gene.

Measurement of CDS1 and CDS2 Protein Levels

Protein extract was mixed and an aliquot used to measure total proteincontent using the Bradford assay. Aliquots containing 5, 10, or 20-μgprotein were then taken for dot blot analysis; the lower protein samplesyielded more consistent data for cds2 while the higher protein samplesworked best for cds1 seeing as cds2 was expressed at much higher levelsthan cds1. The sample was diluted with PBS, transferred into thereservoir of a Bio-Dot SF microfiltration apparatus (Bio-Rad), andfiltered under steady pressure onto a nitrocellulose blotting membrane.The protein-blotted membrane was blocked with 5% fat-free dry milk inPBS containing 0.1% Tween 20 (PBST) and incubated overnight at 4oC withCDS1 antibody (1:1000 dilution) or CDS2 antibody (1:1000 dilution) orbeta-actin antibody. The CDS1 and CDS2 antibodies used were generatedfrom peptide epitopes of the human CDS1 (amino acid sequence:CDDRYGDLDSRTDSDI (SEQ ID NO: 10)) and CDS2 (amino acid sequence:CDKESESEAKVDGETA (SEQ ID NO: 11)), respectively. The reactivity andspecificity of the antibodies were first validated before use (seeresults in FIG. 1). Membranes were washed 3× with PBST and thenincubated with HRP-conjugated secondary antibodies (1:10,000) for 1 h atroom temperature. Visualization was performed by use of Amersham ECLplus Western blotting detection system (GE healthcare, Buckinghamshire,UK). Chemiluminescence images were analyzed by densitometry using theKodak ImageStation 4000 MM. CDS1 and CDS2 densitometric values werenormalized against beta-actin and the data are expressed as relativedensitometric units×10³.

Generally, GAPDH and/or beta-actin were used as standards. Precautionsincluding assessment of RNA quality, parallel analysis of control anddepression samples, and triplicate analyses of each sample were appliedas in previous studies of other postmortem brain transcripts (73-75).

Data Analysis

Data from the various experiments were normalized relative to therespective control or basal measurements, and then pooled for analysis.Data were tested by an appropriate analysis of variance (ANOVA) usingSPSS software (SPSS, Chicago, Ill., USA). Where warranted, the ANOVAswere followed by post hoc analyses using the Dunnett test to comparevarious treatment means to their respective controls. Statisticalcomparisons were considered significant at p<0.05 or better.

After data collection, sample codes were broken to reveal the diagnosticcategory of each sample and permit data analysis. Data were collated andmanaged in SPSS software (SPSS Inc, Chicago, Ill.). Quantitative datafor each dependent variable (CDS1 mRNA, CDS1 protein, CDS2 mRNA, CDS2protein) were subjected to an independent samples (IS) t-test to comparethe control versus depression measurements for each tissue. Further,each analyte was examined by analysis of covariance to exclude (oraccount for) the possible contributions of the various demographicfactors (e.g., sex, race, pmi) to the study variables. Analyses withsubcategories of independent variables, such as diagnosis andantidepressant exposure (e.g., Con, Dep-NoAD, Dep+AD) were alsoexplored. For all analyses, comparisons between mean values wereconsidered statistically significant at p<0.05 or better.

EXAMPLE 2 Chemically Diverse Antidepressant Agents IncreaseCDP-diacylglycerol Production

Diacylglycerol released from phospholipid breakdown is normally rapidlyphosphorylated to produce phosphatidic acid. In the presence of[³H]cytidine-labeled CTP, however, the phosphatidic acid is converted toradiolabeled CDP-DG, which can be extracted and separated away fromother labeled metabolites and subsequently quantified. Rat braincerebrocortical, hippocampal, and striatal slices prelabeled with[³H]cytidine were incubated with various concentrations of selectedantidepressant agents in the presence of LiCl, and the yield ofCDP-diacylglycerol analyzed. Data for each drug were separately analyzedbefore they were normalized and collated together for graphicalpresentation as shown.

The classical antidepressants imipramine and desipramine, the selectiveserotonin reuptake inhibitors fluoxetine and paroxetine and the atypicalagents maprotiline and nomifensine each significantly anddose-dependently enhanced the accumulation of [³H]CDP-DG in rathippocampal, prefrontal cortical, and striatal slices. Whileconcentrations ranging from 0.1 to 1000 μM were tested, only thoseconcentrations lying between the minimal that gave statisticallysignificant effects for any agent (1-3 μM) and the maximally effectiveconcentrations (100-500 μM) are shown. Statistically significant effectswere obtained at concentrations as low as 3-10 μM in the hippocampus orprefrontal cortex, while maximal effects were achieved at the 100 μMconcentration of fluoxetine or 300 μM concentrations of most otheragents. For all agents, test concentrations greater than 300 μM resultedin CDP-DG effects that were either statistically similar to, orsignificantly lower than, the effects observed at 100 μM for fluoxetineor 300 μM for the other agents. This reduction in response withincreasing concentration after attaining maximal responses was moreapparent with the SSRIs, fluoxetine and paroxetine, than with thetricyclic agents.

Similar to the effects exhibited by the classical antidepressant agents,the monoamine oxidase inhibitors (MAOIs), phenelzine and hydralazine,produced robust effects on CDP-DG accumulation in frontal cortex slices,while tranylcypromine had statistically significant but modest effects.While the effects of phenelzine achieved significance at 1 μM(Dunnett's, p<0.01), those of hydralazine became significant at the 10μM and higher concentrations.

Also, a range of other psychotropic compounds were tested to estimatethe extent to which the CDP-DG response may characterize compounds withantidepressive activity. No significant effects or concentration-relatedeffects on CDP-diacylglycerol accumulation in rat cerebrocortical sliceswere observed with the MAOIs pargyline, selegiline, or quinacrine, theantipsychotics chloropromazine, haloperidol, sulpiride, andflupenthioxol and the anticonvulsants or anxiotytics phenobarbital,phenytoin, diazepam, nitrazepam, benztropine, phenylephrine,chlordiazepoxide, and hydroxylamine. Agents were tested at multipleconcentrations ranging from 0.1-300 μM. Data from up to three separateruns were normalized and pooled for analysis by One-Way ANOVA.

Among the brain regions, the hippocampus appeared to be more sensitive,i.e., greater response magnitudes at lower concentrations, whereas thestriatum gave slightly more robust, i.e., maximally attained, effects.The drug responses were statistically dose-dependent for all effectiveagents in each tissue, but there were noticeable differences in potencyor efficacy among the compounds as shown in the data. Thus, diverseantidepressant agents can acutely induce CDP-DG synthesis indepression-relevant regions of the rat brain

EXAMPLE 3

Antidepressant-Induced CDP-diacylqlycerol Formation Translates intoIncreased Phosphoinositide Synthesis

To test if the antidepressant-enhanced CDP-DG translates into increasedsynthesis of the PIs, brain slice preparations were labeled with [³H]inositol and incubated in the presence of various antidepressant agents.Results of the subsequent uptake and conversion of [³H] inositol intoinositol phospholipids are provided herein. Imipramine, desipramine,fluoxetine, paroxetine, and maprotiline each significantly increased[³H]inositol labeling of PIs in the tested brain regions. MAOIs thatwere effective in inducing CDP-DG production also showed enhancedeffects on PI resynthesis, whereas other MAOIs that were ineffective onCDP-DG were equally ineffective in increasing PI resynthesis. Thus, theincreased mobilization or recapture of CDP-DG by the antidepressantagents translates into increased regeneration of PI signalingsubstrates.

EXAMPLE 4 Antidepressant Agents Generally Enhance Inositol PhosphateAccumulation

To test if resynthesized PIs might contribute to enhanced IPaccumulation, agents tested for effects on CDP-DG were also tested in astandard IP assay. Across a concentration range of 3-300 μM, imipramine,desipramine, fluoxetine, paroxetine, and maprotiline significantly anddose-dependently stimulated the accumulation of IPs in each brainregion. Significant drug effects were generally evident atconcentrations of 3-10 μM, while maximal effects were observed at100-300 μM. With imipramine tested in the hippocampus and striatum asthe only possible exceptions, test concentrations greater than 300 μMresulted in IP effects that were either statistically similar to, orsignificantly lower than, the effects observed at the corresponding 300μM concentration. In general, drug concentrations greater than 300-500μM were associated with IP levels that were significantly lower thaneffects at 100-300 μM concentrations, possibly reflecting toxicity fromexcessive stimulation.

EXAMPLE 5 Antidepressant-Induced CDP-diacylglycerol Formation PartiallyDepends on Phosphoinositide Hydrolysis

Phosphoinositide hydrolysis is a major source, but not the only possiblesource, of diacylglycerol in the cell. To estimate the extent to whichantidepressant-enhanced CDP-DG may derive from PI breakdown, PIhydrolysis was blocked and the consequent effects on the ability ofantidepressant agents to induce CDP-DG accumulation were measured.First, the effects of the general PI metabolism inhibitor, neomycin, wastested against the maximally effective concentrations of the selectedantidepressant agents. Neomycin concentration-dependently blocked theeffects of imipramine, desipramine, fluoxetine, paroxetine, maprotiline,or nomifensine on CDP-DG production, PI resynthesis or IP accumulationin hippocampal or prefrontal cortical brain slices. Increasingconcentrations of neomycin produced complete blockade of both CDP-DG andPI responses.

Next, the effects of the selective PLC inhibitor, U73122, were tested onthe drug responses. U73122 by itself did not significantly alter basalCDP-DG production or IP accumulation, although a slight increase in IPwas consistently noted. At concentrations ranging from 0.1 to 10 μM,U73122 significantly reduced, but was unable to completely block,antidepressant drug effects on CDP-DG production. Conversely, the PLCinhibitor completely blocked IP stimulation by 100 μM fluoxetine or 300μM concentrations of imipramine, paroxetine, maprotiline, or nomifensinein hippocampal or cortical slices.

To validate the effects of U73122, the compound was tested against theaction of SKF38393, a D₁ receptor agonist that is known to induce PIhydrolysis in these brain tissues (57,70). SKF38393-induced IPaccumulation was blocked by U73122 with similar efficacies to theinhibition of the antidepressant responses. Moreover, U73123, an analogof U73122 that is ineffective in blocking PLC activity, was withouteffect on any of the CDP-DG or IP responses (data not shown). Theeffects of the SSRIs fluoxetine and paroxetine were more sensitive toinhibition by U73122 than the effects of the tricyclic agents.

EXAMPLE 6 Lithium is Not Required for Antidepressant Drug Effects onCDP-diacylglycerol

These experiments were designed to compare antidepressant drug effectson the IP and diacylglycerol arms of the inositol cycle. Thus, it wasnecessary to include LiCl in all test incubations. Li⁺ is needed toblock inositol monophosphatase and thereby enable the accumulation ofreleased IPs to measurable levels. Selected antidepressant agents weretested for effects on CDP-DG in the absence or presence of 5 mM LiCl todetermine if Li⁺ must be present to demonstrate antidepressant drugeffects on CDP-DG. LiCl did not significantly enhance or inhibitantidepressant drug-induced CDP-DG production, implying that thepresence of Li⁺ is not necessary to demonstrate the enhancing effects ofantidepressant agents on CDP-DG production.

EXAMPLE 7

Antidepressants Elicit Relatively Greater Stimulation ofCDP-diacylglycerol Production than IP Formation

To determine if antidepressant agents exert differential effects onCDP-DG production compared to PI hydrolysis, the ratios of CDP-DGproduction relative to the IPs (CDP-DG/IP ratio) in correspondingtreatment conditions were examined. The ratios were calculated from thedata herein and the results are provided. With each antidepressantagent, the CDP-DG/IP ratios increased significantly with increasing drugconcentrations. This was true for different classes of drugs, includingthe MAOIs phenelzine and hydralazine.

EXAMPLE 8 Monoamine Receptor Agonists Exert Divergent Effects onCDP-diacylglycerol

To determine which, if any, of the endogenous monoaminergic systems mayshow similar profiles of CDP-DG/IP effects, agonists that act directlyat PLC-coupled monoaminergic receptors: α-methylserotonin (5HT₂serotonergic), carbachol (muscarinic cholinergic), SKF38393 (D₁-likedopaminergic), and phenylephrine (alpha-adrenergic) were used.Corresponding CDP-DG ratios were calculated as for the antidepressantagents. As shown, α-methylserotonin, carbachol, phenylephrine orSKF38393 significantly increased IP accumulation and CDP-DG productionin frontal cortex or hippocampal tissues. Carbachol failed to increasePI synthesis, SKF38393 significantly enhanced PI synthesis, while theother two agents had significant but relatively small effects on PI. Theratios of CDP-DG production relative to IP accumulation are providedherein.

With both carbachol and phenylephrine, there was a dramatic decrease inthe CDP-DG/IP ratio. While the ratio did not decrease as much fora-methylserotonin, there was no concentration-related increase either.Conversely, SKF38393 increased CDP-DG/IP ratios significantly and in amanner similar to the antidepressants. Indeed, even the ratios of CDP-DGrelative to PIs or the combination of both inositol derivatives(CDP-DG/IP&PI) were significantly enhanced. Thus, agonists at the directPLC-coupled monoamine receptors showed parallel and correspondingchanges between CDP-DG and the inositides, except for the dopamineagonist which, like the antidepressants, induced proportionately greaterproduction of CDP-DG relative to inositide derivatives.

EXAMPLE 9

Antidepressants Enhance Methylserotonin-Stimulated IP Accumulation in[³H]inositol-Prelabeled Tissues

Brain hippocampal, frontal cortical, and striatal slices were labeledwith [³H]inositol in the presence of various concentrations of selectedantidepressant agents and, after washing the tissues, aliquots of theslices were incubated with indicated concentrations of α-methylserotoninfor an additional 60 min. The levels of accumulated [³H]IPs, assayed byDowex ion exchange chromatography, are provided herein for thehippocampus, frontal cortex and striatum. By itself, α-Me5HT inducedsignificant increases in IP accumulation; these effects, however, weresignificantly enhanced in tissues that had been prelabeled withtritiated inositol in the presence of the antidepressant agentimipramine, desipramine, fluoxetine paroxetine, and maprotiline.

The effects of α-methylserotonin were further concentration-dependentlyenhanced in hippocampal tissues that were prelabeled in the presence ofeach antidepressant agent (p<0.001), in frontal cortex tissuesprelabeled in the presence of imipramine and desipramine (p<0.02) andthe other agents (p<0.001 each), and in striatal tissues prelabeled inthe presence of fluoxetine, paroxetine, and maprotiline (p<0.001).α-Methylserotonin effects, however, were not significantly enhanced instriatal tissues prelabeled in the presence of imipramine or desipramine(p>0.05). In all tissues where there were significant effects ofantidepressant agents, there were significant interactions between theconcentrations of antidepressant agent used and the concentrations ofthe 5HT2 agonist tested. Hence, the concentration-related effects ofa-methylserotonin were maintained, but accentuated, in tissuesprelabeled under the influence of the antidepressant agents.

The zero α-methylserotonin data represent tissues that had beenprelabeled in the presence of the indicated concentrations ofantidepressant agents, further incubated alongside theα-methylserotonin-tested tissues, and subsequently analyzed for thebasal content of inositol phosphates. In these tissues that did notreceive α-methylserotonin, there were generally increased levels of IPswith increasing concentrations of the antidepressant agents.

Thus, mere prelabeling of the tissues in the presence of antidepressantagents led to increased accumulations of inositol phosphates even in theabsence of exogenous 5HT2 receptor stimulation. These effects werestatistically significant for all agents at at least their highesttested concentrations in each brain region (ANOVA, p<0.001 in eachcase). The net effects of the combined exposure to antidepressant andα-methylserotonin were not significantly different from the sum of theseparate effects of antidepressant and α-methylserotonin, thussuggesting an additive mechanism of interaction between AD andα-methylserotonin treatments.

EXAMPLE 10 Effects of 5HT2 Receptor Blockade on Drug-Induced IPAccumulation

To examine the extent to which the enhancing effects of antidepressantagents on inositol phosphate accumulation were dependent on postsynaptic5HT2 receptor stimulation, each antidepressant was challenged with arange of concentrations of the 5HT2 receptor-selective antagonist,LY53857 (71-72). The antagonist was added after the prelabeling phase,but 15 min prior to the addition of a-methylserotonin to the incubatingslices. LY53857 did not significantly alter the basal levels of IPs orPIs, but increased CDP-diacylglycerol by 20% at the 0.1 μMconcentration. The 5HT2 antagonist completely blocked the IP responsesto each of the AD agents (p<0.001 in each case). PI labeling andCDP-diacylglycerol responses were statistically significantly inhibited(p<0.05 or better) for all drugs; however, only the inhibition of PIlabeling induced by fluoxetine and paroxetine was substantial.

While the results shown are for the hippocampus, similar observationswere made in frontal cortex tissues. With regard toantidepressant-enhanced phosphatidylinositol resynthesis, 5HT2 receptorblockade significantly inhibited the effects of the SSRIs fluoxetine andparoxetine, but the effects of the tricyclic agents imipramine anddesipramine or those of maprotiline were only partially, thoughsignificantly, reduced. Moreover, antidepressant drug effects onCDP-diacylglycerol were only minimally, though statisticallysignificantly, inhibited by the 5HT2 antagonist. Thus,antidepressant-facilitated release of IP second messengers requiresintact 5HT2 receptor function, whereas antidepressant drug effects onCDP-diacylglycerol or phosphatidylinositol labeling may facilitate, butnot depend on, postsynaptic 5HT2 receptor signaling.

EXAMPLE 11 Effects of Blocking Endogenous PI Metabolism onAntidepressant-Induced Behavioral Effects in the Forced Swim Test

Imipramine was first tested by both the conventional regimen using oneacclimation with three-point drug administration and the currentmodification using two acclimations with two-point drug administration,of the forced swim test. Either method produced significant dose-relatedeffects for imipramine on immobility behavior (p<0.001 for eachdataset). Immobility times were slightly higher in the modified testthan in the conventional test (p<0.05). Most significantly, thevariability in the data was much reduced in animals that underwent twoacclimation sessions; hence, the mean coefficient of variation was2.6-fold lower in the modified test than in the conventional approach.

Following pretreatment with saline (controls) or neomycin, animals weresubjected to the forced swim test in the presence or absence of selectedantidepressant agents. Imipramine, fluoxetine, and maprotiline eachinduced significant and dose-dependent reductions of immobility times inthe forced swim test, and these effects were completely reversed inanimals that received neomycin pretreatments. Yet, neomycin by itselfdid not significantly alter basal immobility times in any of theexperiments. SKF38393 was tested in the absence and presence ofneomycin. SKF38393 significantly decreased immobility times similar tothe effects of the clinical antidepressant agents and this action wasblocked by neomycin. Apparently, the acute behavioral effects of thedrugs in at least the forced swim test depend on intact functioning ofbrain PI systems.

EXAMPLE 12

Specific Immunoreactivity of Generated cds1 and cds2 Antibodies

Rabbit antibodies raised against human cds1 and cds2 peptides (identicalin sequence to rat cds1 and cds2) respectively immunoreacted against thespecific peptides, and detected the respective proteins in ratcerebellar or testicular tissue extracts (these tissues express highlevels of cds1/cds2) (FIG. 1). HEK293 cells transfected with cds1 orcds2 plasmids showed high reactivity to the respective antisera incomparison with relatively weaker reactions of the antisera againstuntransfected HEK293 cells. Subcellular fractionation experiments showedhighest reactivity to cds1 in the microsomal fraction while the highestreactivity to cds2 was present in the mitochondrial cellular fraction(rat data shown in FIG. 1 for cds1 reflect microsomal fraction and forcds2 the mitochondrial fraction, whereas human brain or blood sampleswere not fractionated).

EXAMPLE 13

CDS1 and CDS2 mRNA and Protein Levels in Postmortem Human Brain ofControl and Depression Subjects

Quantitative PCR analysis of cds1 and cds2 transcripts in medialprefrontal cortex samples of 28 matched pairs of control (Con) anddepression (Dep) subjects were conducted. CDS protein levels were alsoanalyzed by semiquantitative Western Blotting of triplicate tissuelysates from 16 pairs of qualified subjects. Statistical analysesyielded the following results:

CDS1 and CDS2 protein or mRNA expression levels were consistent with theNormal distribution for both the Con and Dep subjects (K-S test). Onthis basis, robust parametric approaches were used for inferentialanalysis of the data.

The mRNA levels of cds1 were significantly correlated with cds2(p<0.001), as well as the cds1/cds2 ratio (p<0.01) when examined acrossall subjects or separately for each diagnostic group. This could reflectsample variability, in which case using the ratio data can correct forsome of that variability, or it may suggest that cds1 and cds2 areco-regulated.

Expression levels of cds1 or cds2 mRNA or protein did not correlate withsubject age (15-50 y) or postmortem interval (3-33 h). An IndependentSamples (IS) t-test showed no significant differences in cds1 or cds2mRNA levels when compared on sex (18 male v. 24 female) or race (8African v. 34 caucasian), although the data trended toward lower cds1(p=0.167) or cds2 (p=0.146) in the older group. Protein levels of cds1appeared significantly higher in the African v. Caucasian subjects(p<0.05). With the possible exception of the race observations, thedemographic factors may not play a significant role in determining thebrain cortical levels of cds transcripts. This increases the strengthand clinical significance of any associations that may be found betweencds mRNA levels and a diagnosis of depression. Mean cds1 mRNA levels inthe Dep group (0.0284±0.0037) were significantly lower thancorresponding levels in the Con group (0.0417±0.0044) when compared byPaired Samples (PS) t-test (p<0.01) or IS t-test (P<0.03) (FIG. 2A).Similar results were obtained for cds1/cds2 ratio data. Mean expressionlevels of cds2 mRNA, however, were not significantly different betweenCon (0.2642±0.0251) and Dep (0.2447±0.0216) samples when compared by PS(p>0.226) or IS (P>0.561) t-tests.

Similar to the cds1 mRNA results, mean cds1 protein levels weresignificantly lower in the Dep group (0.70612±0.058057) compared to theCon group (0.94494±0.059317) by IS t-test (p<0.007, N=16) (FIG. 2B). Thefinding that cds1 levels were significantly decreased in the Dep groupimplies that cds1, but not necessarily cds2, is a biosignature indepression.

EXAMPLE 13

Effects of Antidepressant Medication Use on cds1 mRNA and Protein Levels

Based on information from autopsy or medical history reports, subjectswere identified who had been on antidepressant (AD) medication prior todeath (history) or at death (autopsy). These subjects were thenclassified based on Diagnosis-AD medication as follows: Controlsubjects-No AD, Dep-Noncurrent AD, and Dep+Current AD. A One-Way ANOVArevealed significant main effects (p<0.03 for cds1mRNA; p<0.03 for cds1protein; p<0.005 for cds1/cds2 mRNA ratio). Subsequent posthoc Dunnettanalyses showed significant differences (p<0.02) between the higher meancds1 mRNA values in Con-No AD subjects (0.0417±0.0044) and lower meancds1 mRNA values in the Dep-Noncurrent AD group (0.0256±0.0031) (FIG.2A). CDS ratio data gave similar conclusions, but with highersignificance levels. The posthoc analyses for cds1 protein showed nosignificant differences between any pair of groups, possibly because ofthe low sample sizes involved (see graph); nevertheless, there weretrends toward the direction of the mRNA data (FIG. 2B).

Interestingly, Dunnett analysis of the cds1 mRNA data indicated nodifference between control subjects and depression subjects who werecurrently using antidepressant medications (mean=0.0403±0.0095; p>0.78).Antidepressant drugs in the current AD drug users may have “normalized”the decreased cds1 levels seen in nonmedicated depressed subjects.

EXAMPLE 14

CDS1 and CDS2 mRNA and Protein Levels in Circulating Peripheral Blood ofNormal Human Volunteers

Each subject was tested on five separate occasions and each sample drawnwas tested in duplicate. The mean CDS1 values obtained in Replicate1versus Replicate2 were 2.014±0.261 and 2.076±0.278 (N=5), while for CDS2these values were 564.7±91.4 and 762.2±98.8 (N=5), respectively. Thereis no statistically significant difference between the CDS1 (p>0.68) orCDS2 (p>0.16) replicates, while there is a highly significant Pearsoncorrelation between the replicate cds1 values (p<0.001), thus supportingthe high reproducibility of the assay method.

Reproducibility of Analyte Measurements

Data obtained across the five test sessions were analyzed by RepeatMeasures ANOVA which showed no significant difference in mean valuesacross the experimental sessions for CDS1 (p>0.134) or CDS2 (p>0.494).Thus, for normal subjects, the assay is reproducible across testsessions and the cds values remain stable over this time period.

Relationship Between CDS1 and CDS2 Expression Levels

Mean levels of cds1 (2.045±0.259) and cds2 (663.5±67.7) (N=5) weresignificantly different (p<0.001), and their relative abundancecorresponds well with the preceding observations in human postmortembrain tissues.

CDS1 Protein Expression

Mean cds1 protein expression was 2.132±0.089 relative to beta-actinprotein levels. This method and the findings provide an overallindication of normal blood CDS1 (and cds2) expression in living humansubjects. Both the mRNA and protein levels of cds1 were measuredreproducibly in microliter aliquots of human blood samples. Overall, itis remarkable that such relatively high levels of cds1 mRNA and proteincould be measured reproducibly in microliter samples (technically fivedrops) of whole human blood.

EXAMPLE 14 Discussion and Conclusion

The present invention found decreased expression levels of CDS1 but notnecessarily CDS2 in a depression-relevant region of the frontal cortexfrom depressed subjects compared to normal controls. A well-correlatedtrend of increasing CDS1 expression was found in depression subjectsthat were on antidepressant medication around the time of death,implying that the drug treatments might have been working to restore thenormal levels of CDS1 expression and/or activity. Furthermore, blockadeof downstream CDP-diacylglycerol activity prevented the therapy-relevantbehavioral effects of the antidepressant drugs. The data are noteworthyin establishing a reproducible assay for the quantification of CDSexpression in relatively small amounts of whole human blood, thushinting at the potential clinical applicability of the overall results.

The CDP-diacylglycerol (CDG)/CDP-diacylglycerol synthase (CDS) system isubiquitous and plays unique roles in cellular function. The enzymaticactivity of CDS exists as two protein isoforms, CDS1 and CDS2, either ofwhich produces CDP-diacylglycerol, a nucleolipid. The nucleolipidproducts of CDS may have arachidonoyl or stearoyl derivatives at theC-1/C-2 position of the glycerol moiety. Studies in nonhuman speciesindicate that CDS1 produces primarily2-arachidonoyl-[CDP-diacylglycerol] which is utilized for the productionof phosphatidylinositol for downstream multipartite metabotropicsignaling cascades; conversely, the primary products of CDS2 arecomparatively saturated derivatives of CDP-diacylglycerol (e.g.,2-stearoly-[CDP-diacylglycerol]) that are utilized for the synthesis ofphosphatidylglycerol and ultimately cardiolipin biosynthesis. It hasbeen suggested that CDS1 is localized primarily to the endoplasmicreticulum, whereas CDS2 is localized primarily in mitochondria. Theseobservations are consistent with the differential effects of depressiveillness on CDS1 versus CDS2, and also hint at the possibility thatseparate functions may be found for CDS2 in physiological orpathological conditions such that drugs or diagnostics targeting CDS1may not necessarily impact on the biological functions of CDS2.

The CDS/CDG model of depression or antidepressant drug mechanisms is thefirst to present a potentially unifying molecular explanation for thebeneficial clinical effects of the wide variety of available ADmedications. An upstream action at the level of CDG synthesis andsignaling could potentially explain many previously observed effects ofAD drugs via the downstream cascades that would be modulated by enhancedCDG: enhanced PI/PLC leading to PKC/CamK activation, or increasedPI/PI3K leading to Akt/GSK3_ or mTOR regulation. For instance, PI is akey substrate for the PI3K signaling pathway. Earlier reports showedthat PI3K signaling is relevant to the induction of neurogenesis orneuronal survival and plasticity. Recent reports demonstrated that theactivity of PI3K and its downstream target, Akt, are compromised inpostmortem brain tissues of depressed suicide victims (76-77). Furtherdownstream, these systems crosstalk to regulate CREB phosphorylation andBDNF signaling—processes that have been implicated in neurogenesis andAD mechanisms.

Thus, it is conceivable that an early action of AD agents to enhance themobilization of CDG could lead to coordinate effects on multiplesignaling systems, which might then explain the various molecular andfunctional effects of the drugs. By the proposed CDS/CDG model, an ADagent would minimally possess the ability to enhance CDG productionand/or increase synaptic levels of brain monoamine neurotransmitters:serotonin (which does not by itself increase CDG but robustly generatesdownstream second messengers from CDG-derived PI), norepinephrine (whichmodestly enhances CDG resynthesis as well as generate PI signalingmessengers) or dopamine (which robustly induces CDG synthesis andmoderately increases PI/PLC signaling). A clear advantage, of course,would exist for those agents that possess dual abilities to raisesynaptic monoamines and increase CDG synthesis; this may explain whymany clinically effective AD medications such as imipramine,desipramine, maprotiline, fluoxetine, paroxetine, and phenelzinegenerally have these dual properties. These are innovative ideas thatcould open a new path through the formidable task of exploring thecauses and clinical course of depressive illness and its treatment. Thepresent study focusing on the CDS enzymes has extended prior work withCDG, the enzyme product, revealing consistent modulations of the CDS/CDGsystem in human depression.

The present report establishes CDS (particularly CDS1) as a correlate ofdepressive illness, thus advancing the idea to apply the levels of CDS1enzymatic activity, protein, messenger RNA, and/or gene polymorphisms ina person's blood or other tissue sample to determine the likelihood(decreasing cds1 levels with repeated sampling), presence/severity(critically low cds1 levels as compared to previous levels or normativestandards), or improvement/relief (recovery of cds1 levels abovecritical functional points) of major depression in humans. Thebiomedical literature contains numerous examples of correlations betweenbrain and blood molecular or neurochemical indices of depression. Forexample, imipramine binding levels in blood platelets correlate withimipramine binding levels in brain tissue. Therefore, demonstration ofdecreased CDS1 mRNA (and protein) in the brain will correlate withsimilar decreases in the blood. Thus, measurements of CDS1 levels(absolute or relative to cds2) in the blood should reproducibly indexcorresponding brain measures and therefore serve as a reliable index forthe diagnosis and prognostication of major depression. Indeed, theability of a drug or other treatment to enhance CDS activity (viaincreased expression, decreased inactivation, or enhanced enzymeactivation) could become indicative of ultimate antidepressant efficacy.

Major depression is currently diagnosed, and its treatment monitored, bysubjective personal interview. There is currently no objective molecularor biochemical diagnostic test for depression, nor is there a test thatindicates the severity or improvement of the disorder. The presentinvention holds promise for the development of a series of tests thatcould signal susceptibility to depression (if functionally relevantpolymorphisms are detected in the person's CDS1 gene), an impendingepisode of depression (if repeated assays show decreasing levels ofCDS), the severity of existing depression (if the levels of CDS fallbelow critical levels), or efficacy of treatment (if repeated samplingsshow improving—increasing—levels of CDS). Levels of CDS may refer to theenzymatic activity of CDS, the protein concentration of CDS (measured bystandard assays such as electrophoresis, ELISA, mass spectrometry, etc),the mRNA concentration of CDS (measured by standard procedures such asquantitative PCR), or the polymorphism status of the CDS1 gene (measuredby relevant genotyping assays).

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Any publications mentioned in this specification are indicative of thelevels of those skilled in the art to which the invention pertains.Further, these publications are incorporated by reference herein to thesame extent as if each individual publication was specifically andindividually incorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1. A method of diagnosing a depressive disorder in a test subject,comprising: determining CDP-diacylglycerol synthase 1 enzyme activity orCDP-diacylglycerol synthase 1 expression level in the test subject; andcomparing said enzyme activity or expression level to enzyme activity orexpression level of CDP-diacylglycerol synthase 1 in a control subject,wherein a statistically lower CDP-diacylglycerol synthase 1 enzymeactivity or CDP-diacylglycerol synthase 1 enzyme expression level in thetest subject indicates the test subject has a depressive disorder. 2.The method of claim 1, wherein the expression level is determined bymeasuring a CDP-diacylglycerol synthase 1 protein.
 3. The method ofclaim 1, wherein the expression level is determined by measuring a ratioof a CDP-diacylglycerol synthase 1 protein to a CDP-diacylglycerolsynthase 2 protein.
 4. The method of claim 1, wherein the expressionlevel is determined by measuring a CDP-diacylglycerol synthase 1 mRNA.5. The method of claim 1, wherein the expression level is determined bymeasuring a ratio of a CDP-diacylglycerol synthase 1 mRNA to aCDP-diacylglycerol synthase 2 mRNA.
 6. The method of claim 1, whereinthe enzyme activity or expression level of CDP-diacylglycerol synthaseis determined in blood, brain tissue, testis tissue, eye tissue orsmooth muscle tissue.
 7. The method of claim 1, wherein the depressivedisorder is major depression, unipolar depression, bipolar depression,reactive depression, endogenous depression, or dysthymic disorder. 8.The method of claim 1, wherein a CDP-diacylglycerol synthase 1 proteinlevel in said test subject that is about 75% or lower than aCDP-diacylglycerol synthase 1 protein level in the control subjectindicates that the test subject has a depressive disorder.
 9. The methodof claim 1, wherein a CDP-diacylglycerol synthase 1 mRNA level in saidtest subject that is about 68% or lower than a CDP-diacylglycerolsynthase 1 mRNA level in the control subject indicates that the testsubject has a depressive disorder.
 10. The method of claim 1, wherein aratio of a CDP-diacylglycerol synthase 1 mRNA to a CDP-diacylglycerolsynthase 2 mRNA in said test subject that is about 73% or lower than aratio of a CDP-diacylglycerol synthase 1 mRNA to a CDP-diacylglycerolsynthase 2 mRNA in the control subject indicates that the test subjecthas a depressive disorder.
 11. A method of predicting therapeuticefficacy of an antidepressant drug in a subject with a depressivedisorder, comprising: determining CDP-diacylglycerol synthase 1 enzymeactivity or expression level of CDP-diacylglycerol synthase 1 in thesubject; administering an antidepressant drug to the subject; andmonitoring CDP-diacylglycerol synthase 1 enzyme activity or expressionlevel of CDP-diacylglycerol synthase 1 in the subject afteradministration of the antidepressant drug to the subject, wherein astatistically higher enzyme activity or expression level afteradministration of the antidepressant drug to the subject indicates thatthe antidepressant drug has therapeutic efficacy for the subject. 12.The method of claim 11, wherein the antidepressant drug is a tricyclicantidepressant, a selective serotonin reuptake inhibitor, an atypicalantidepressant or a synthetic antidepressant compound that increases anenzyme activity or expression level of a CDP-diacylglycerol synthase 1in a depression-relevant brain tissue or blood platelets.
 13. The methodof claim 12, wherein the tricyclic antidepressant is desipramine orimipramine and the selective serotonin reuptake inhibitor is fluoxetineor paroxetine.
 14. The method of claim 11, wherein the depressivedisorder is major depression, unipolar depression, bipolar depression,reactive depression, endogenous depression, or dysthymic disorder. 15.The method of claim 11, wherein the expression level is determined bymeasuring CDP-diacylglycerol synthase 1 protein.
 16. The method of claim11, wherein the expression level is determined by measuring a ratio ofCDP-diacylglycerol synthase 1 protein to CDP-diacylglycerol synthase 2protein.
 17. The method of claim 11, wherein the expression level isdetermined by measuring CDP-diacylglycerol synthase 1 mRNA.
 18. Themethod of claim 11, wherein the expression level is determined bymeasuring a ratio of CDP-diacylglycerol synthase 1 mRNA toCDP-diacylglycerol synthase 2 mRNA.
 19. The method of claim 11, whereinthe enzyme activity or expression level of CDP-diacylglycerol synthaseis determined in blood, brain tissue, testis tissue, eye tissue orsmooth muscle tissue.
 20. A method of identifying a compound effectiveto treat or alleviate the symptoms of depression, comprising:determining level of CDP-diacylglycerol synthase 1 enzyme activity orexpression level of CDP-diacylglycerol synthase 1 in a tissue;contacting said tissue with a potential antidepressant compound; anddetermining level of CDP-diacylglycerol synthase 1 enzyme activity orexpression level of CDP-diacylglycerol synthase 1 in the tissue aftercontact with the compound, wherein a statistically higherCDP-diacylglycerol synthase 1 enzyme activity or CDP-diacylglycerolsynthase 1 enzyme expression level in the tissue after treatment withthe compound indicates that the compound has an antidepressant effect.21. A kit for diagnosing a depressive disorder, comprising: an antibodyto CDP-diacylglycerol synthase 1 protein; an antibody toCDP-diacylglycerol synthase 2 protein; and instructions for measurementof said proteins for the diagnosis of said depressive disorder.
 22. Akit for diagnosing a depressive disorder, comprising: forward andreverse primers for CDP-diacylglycerol synthase 1 gene; a probe thathybridizes with CDP-diacylglycerol synthase 1 gene; forward and reverseprimers for CDP-diacylglycerol synthase 2 gene; a probe that hybridizeswith CDP-diacylglycerol synthase 2 gene; and instructions formeasurement of said genes for the diagnosis of said depressive disorder.